The invention relates to a process for purifying 20(S)-camptothecin contaminated by a vinyl-camptothecin derivative.
20(S)-camptothecin (20(S)-CPT) is a natural alkaloid of formula (I) 
wherein R1 denotes ethyl.
20(S)-CPT and its derivatives, being topoisomerase I inhibitors, have tumour-inhibiting properties (e.g., B. C. Giovanelle et al., Cancer Research, 51: 302-3055, 1991; European Patent Applications EP 0 074 256 and EP 0 088 642; U.S. Pat. Nos. 4,473,692, 4,545,880, and 4,604,463; and International Patent Application WO 92/05785).
20(S)-CPT can be obtained as a crude product from the Chinese tree Camptotheca acuminata (Nyssaceae) (M. Wall et al., J. Am. Chem. Soc. 88: 3888-3890, 1966) or from the Indian tree Nothapodytes foetida (nimmoniana) (formerly known as: Mappie foetida Miers) (T. R. Govindachari et al., Phytochemistry 11: 3529-3531, 1972), inter alia.
These crude products, particularly the one obtained from Nothapodytes foetida, contain 20(S)-CPT contaminated by a CPT derivative of formula (I) wherein R1 denotes vinyl (20-vinyl-CPT).
Traditionally, the crude products are purified by complex chromatographic methods or by converting the camptothecin into the aqueous phase and eliminating impurities by extraction with water-insoluble solvents (e.g., WO 94/19353). However, contamination by 20-vinyl-CPT cannot be efficiently dealt with by these methods.
The problem of the present invention is therefore to provide a process which allows the 20(S)-CPT starting product to be purified without using complex chromatographic methods.
Surprisingly, it has been found that 20(S)-CPT can be virtually completely freed from contamination with 20-vinyl-CPT by first treating the starting material with an aqueous base, hydrogenating and subsequently acidifying it and then isolating the product.
The invention thus relates to a process for purifying 20(S)-camptothecin which comprises the following steps:
(a) combining an aqueous base and a starting material containing 20(S)-camptothecin, thereby converting the lactone ring of the 20(S)-camptothecin into a carboxylate salt;
(b) hydrogenating the resulting mixture in the presence of a transition metal catalyst;
(c) acidifying the aqueous phase, thereby forming camptothecin crystals;
(d) adding a polar aprotic solvent; and
(e) separating off the camptothecin crystals.
The invention further relates to a process for preparing 20(S)-camptothecin of formula (I) wherein R1 denotes ethyl, from 20-vinyl-camptothecine of formula (I) wherein R1 denotes vinyl, which comprises the following steps:
(a) combining an aqueous base and the starting material containing 20(S)-camptothecin, forming a compound of formula (II), 
xe2x80x83wherein: R1 denotes vinyl, and Met denotes a metal;
(b) hydrogenating the resulting mixture in the presence of a transition metal catalyst;
(c) acidifying the aqueous phase to form camptothecin crystals;
(d) adding at least one polar aprotic solvent; and
(e) separating off the camptothecin crystals.
The term xe2x80x9cstarting material containing camptothecinxe2x80x9d as used above and hereinafter refers to a contaminated material containing 20(S)-CPT, crude camptothecin, camptothecin-containing plant extracts, synthetic camptothecin, derivatives of camptothecin as described, for example, in International Patent Application WO 92/05785, or reaction products containing camptothecin.
Preferably, the starting material is a natural crude product which is obtained in particular from Nothapodytes foetida. As a rule, it is a mixture of the compound of formula (I) wherein R1 denotes ethyl, and the compound of formula (I) wherein R1 denotes vinyl. It generally contains 0.9 wt. % to 1.5 wt. %, preferably 1.0 wt. % to 1.4 wt. % of the vinyl compound. In addition, the starting material may contain other camptothecin derivatives such as, for example, 9-methoxy-CPT, 10-methoxy-CPT, 11-methoxy-CPT, 10-hydroxy-CPT, and 11-hydroxy-CPT. As a rule, the starting material contains up to 1 wt. % of one or more of these additional CPT derivatives, particularly 0.2 wt. % to 0.8 wt. % of 9-methoxy-CPT.
The term xe2x80x9caqueous basexe2x80x9d as used above and hereinafter in connection with step (a) of the purification process according to the invention relates to a base which generates enough hydroxide ions in the aqueous medium, preferably in pure water, to convert the lactone group of the camptothecin derivatives contained in the starting material completely into the corresponding hydroxycarboxylates. Metal hydroxides are preferred, particularly alkali metal or alkaline earth metal hydroxides such as lithium hydroxide, sodium hydroxide, potassium hydroxide or calcium hydroxide. Sodium hydroxide is most preferred.
The metal hydroxide is preferably used in the form of a dilute aqueous solution, preferably in the form of a 1% to 25%, particularly a 3% to 10% aqueous solution. As a rule, sufficient metal hydroxide is used to make the camptothecin derivatives go completely into solution; preferably, 1 mol to 20 mol, more preferably 5 mol to 15 mol, particularly 7.5 mol to 12.5 mol of metal hydroxide are used per 1 mol of starting material.
In step (b) a transition metal catalyst, preferably a heterogeneous transition metal catalyst, particularly platinum, platinum oxide, nickel, palladium or rhodium on a carrier material such as activated charcoal or aluminium oxide is added to the resulting mixture. Palladium on activated charcoal containing 1 wt. % to 15 wt. %, preferably 2 wt. % to 10 wt. %, particularly about 5 wt. % of palladium is particularly preferred.
The quantity of transition metal catalyst is selected so as to ensure total hydrogenation of the vinylic CPT derivative. Preferably, 0.01 to 0.50 parts by weight, particularly 0.02 to 0.10 parts by weight of transition metal catalyst (including carrier materials) are used, based on 1 part by weight of the starting material.
The resulting mixture is subjected to the action of hydrogen gas, preferably at a temperature of xe2x88x9220xc2x0 C. to 100xc2x0 C., particularly 10xc2x0 C. to 40xc2x0 C., most preferably at about room temperature.
The hydrogen pressure is not critical per se; the hydrogenation is preferably carried out at normal pressure or at slightly raised pressure, particularly at 0.9 bar to 5.0 bar, most preferably at about 1 bar.
Under these conditions, hydrogenation is generally complete within 1 to 20 hours, preferably 4 to 15 hours, particularly 6 to 10 hours.
After the hydrogenation has ended, the transition metal catalyst is preferably eliminated by filtration, and the resulting reaction mixture is acidified in step (c). The acidification can be done with an inorganic or organic acid. Preferred acids are inorganic acids such as HCl, HBr, HI, HNO3, H3PO4, H2SO4, or aliphatic carboxylic acids such as acetic acid and trifluoroacetic acid or mixtures of these acids, particularly concentrated hydrochloric acid. Using the chosen acid, the pH is adjusted to 3.0 to 6.0, preferably 3.5 to 5.0, particularly about 4.0 to 4.5. The reaction with the acid is generally carried out at a temperature of 0xc2x0 C. to 100xc2x0 C., preferably 30xc2x0 C. to 80xc2x0 C., particularly 50xc2x0 C. to 60xc2x0 C.
In a particularly preferred embodiment, acidification is carried out with 2 to 20 parts by weight, preferably 4 to 9 parts by weight, particularly 6 to 8 parts by weight of concentrated hydrochloric acid, based on 1 part by weight of starting material.
Under the conditions described, lactonization to form the CPT is generally complete within 10 to 180 minutes, preferably 15 to 60 hours, particularly within about 30 minutes.
The reaction mixture obtained by acidification is generally in the form of a pure suspension. To improve the crystallization in step (d) one or more polar aprotic solvents are added thereto. Suitable solvents of this kind are preferably sulfoxides such as dimethylsulfoxide (DMSO) or amides and urea derivatives of formula 
wherein
R2 denotes hydrogen or a C1-4 alkyl group, particularly hydrogen or methyl;
R3 and R4 independently of each other denote a C1-4 alkyl group, particularly methyl; or
R2 and R3 together denote a xe2x80x94(CH2)mxe2x80x94 or a xe2x80x94NR5xe2x80x94(CH2)nxe2x80x94 group, while
R5 denotes a C1-4 alkyl group;
m is 3 or 4, particularly 3; and
n is 2 or 3,
particularly selected from among N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), N-methylpyrrolidone (NMP), N,N-dimethylethylene urea (DMEU), and N,N-dimethylpropylene urea (DMPU) or mixtures of these solvents, most preferably DMF.
As a rule, 10 to 100 parts by weight, preferably 20 to 80 parts by weight, particularly 30 to 50 parts by weight of the polar aprotic solvent are used, based on 1 part by weight of the starting material used.
The treatment with the polar aprotic solvent may be carried out at any desired temperature. The reaction mixture is preferably stirred at a temperature of 30xc2x0 C. to 120xc2x0 C., particularly 80xc2x0 C. to 100xc2x0 C. and then slowly cooled to ambient temperature.
The CPT crystals thus obtained are easily separated from the liquid phase in step (e), preferably by decanting, centrifuging, spinning, squeezing out or filtration, particularly by filtration.
As a rule, the CPT crystals thus obtained are washed with an alcohol, preferably methanol, ethanol or isopropanol, particularly methanol, and dried.
The advantage of the procedure according to the invention is the high space/time yield and the high yield and purity of the 20(S)-camptothecin produced, which is obtained without any chromatographic purification substantially free from contaminants containing vinyl groups.